Molecular characterisation of very virulent infectious bursal disease viruses in Taiwan

The very virulent infectious bursal disease virus (vv IBDV) RNA in the bursa of Fabricius and spleen from experimentally infected chickens or field samples was detected by in situ hybridisation (ISH) with subsequent reverse transcription (RT)-polymerase chain reaction (PCR) and sequence analysis. The VP 2 gene of vv IBDV was detected by ISH in infected chicken tissues with a cloned digoxigenin (DIG)-labelled cDNA probe. To verify ISH, RT - PCR was used to amplify two 643- and 500-base pair fragments on the VP 2 gene of IBDV in the bursa of Fabricius. With all isolates, two c DNA fragments of 643 and 500 bp long, respectively, were generated as expected and further confirmed the specificity of ISH. Analysis of the hypervariable region (HVR) of the VP 2 gene revealed that a serine-rich heptapeptide SWSASGS located at amino acids 326-332 was conserved in recent Taiwanese strains, and two amino acid substitutions were found in the classical Taiwanese strains at positions 330M and 331W. Three amino acids were unique to the vv strains at positions 222A, 256I and 294I, compared with classical and variant strains. Sequence and phylogenetic analysis showed that the recent Taiwanese strains were closely related, very similar to vv IBDV s from Europe, China, Japan, and Africa, and distantly related to the Taiwanese classical strains.